y membranes according to the manufac turers protocol. Membranes were initially blocked, followed by e posure to cell lysate. After washing, e po sure to biotin conjugated cytokine antibody and HRP conjugated streptavidin, cytokines selleck chem DAPT secretase were detected using standard chemiluminescent methods. The proce dure was performed three times. Determination of MIP 2 e pression by Mesangial Cells MC were initially seeded unto plastic dishes in DMEM supplemented with 10% FBS. Subsequently, cultures were serum starved overnight, fol lowed by incubation with L cysteine or Hcy for 24 hours at 37 C. Cells were harvested and total RNA was isolated by estab lished methods. Following cDNA synthesis, qPCR was performed using an iQ SYBR Green kit.
Detection of MIP 2 Protein in Mesangial cells Cultures were serum starved overnight, followed by incu bation with L Cys or Hcy for 24 hours at 37 C. Subsequently, cells were washed with phosphate buffered saline and harvested under non denaturing conditions by incuba tion with lysis buffer. Following centrifugation, the supernatant was transferred to a fresh microcentrifuge tube and the protein concentration was measured with Bio Rad protein assay reagent. Protein was separated on a SDS PAGE gel. After electroblotting to a nitrocellulose membrane, membranes were incubated with 25 ml of blocking buffer and then over night at 4 C with rabbit polyclonal macrophage inflam matory protein 2 antibody in 20 ml of antibody dilution buffer with gentle rocking. Membranes were washed 3 times with TTBS and then incubated with HRP conjugated anti rabbit secondary antibody in 20 ml of anti body dilution buffer.
After three further TBS washes, the membrane was incubated with ECL Chemilumines cence Reagent and then e posed to ray film. Immune comple es were removed from the membrane by treat ment with stripping buffer. Subsequently, protein loading was assessed by re blotting with anti actin antibody and an HRP conjugated anti rabbit second ary antibody. Pro tein bands were quantified using BioRad Quantity One software AV-951 package. In order to study the effect of kinase inhibitors on MIP 2, MCs were incubated in the presence of Hcy with or without inhibitors U0126, SB203580 and LY294002 for 24 h at 37 C. Subsequently, cells were washed with PBS and har vested under non denaturing conditions by incubation with lysis buffer as described above.
MIP 2 protein was quantified after detection by western blot as described above. Immunofluorescence Microscopy for MIP 2 MCs were initially plated onto sterile two chambered slides e actly as described for other e periments above. After incubation in the presence of Hcy with or without kinase inhibitors, protein inhibitors cells were washed and fi ed. Following PBS washes, cells were permeabilized, washed again with PBS and incubated with blocking solution for 60 minutes at room temperature. The cells were subsequently incubated with rabbit poly clonal MIP 2 antibody constituted in blocking solution. Following PBS washes, cells were